Abstract Bartonella species are slow growing intracellular bacteria that infect a variety of mammalian hosts including companion animals, production animals, wildlife, and humans. Bartonella infection occurs via arthropod vectors, animal bites and possibly inhalation. Bartonellosis is associated with many human diseases including infective endocarditis (IE), infection of the interior of the heart including the heart valves. IE has maintained a high, in- hospital mortality rate of ~20% for over two decades and the number of human cases of Bartonella IE has been increasing at a rate of 2.4% annually in the US since 2005. Bartonella species are well known for the very low- levels of bacterial concentrations associated required for disease. This property presents a significant diagnostic challenge. Current assay strategies are not up to the task of detecting Bartonella in these patients, creating an urgent need for a highly sensitive and rapid assay for the diagnosis of Bartonellosis. Through the work in this proposal, we will leverage our experience in the culturing and molecular detection of bartonellosis to develop an improved Bartonella PCR assay using highly sensitive droplet digital PCR (ddPCR) to allow the more rapid, sensitive, and quantitative diagnosis of Bartonellosis. We will accomplish this goal with the following Specific Aims: Specific Aim 1: Develop and optimize a highly sensitive ddPCR assay for the diagnosis of bartonellosis. Droplet digital (dd) PCR is a relatively new technology with a demonstrated utility in the field of quantitative infectious disease diagnostics. It is far more sensitive than standard PCR methods, enabling the accurate quantification of rare targets, and is less susceptible to interference from other molecules in the sample. Together, these properties make the technology ideal for the detection of Bartonella in blood and heart tissue. Specific Aim 2: Compare ddPCR specificity and sensitivity to the gold standard method of 7-21 day BAPGM enrichment followed by PCR. We will compare ddPCR to our gold standard for Bartonella detection regimen: the culture of blood in BAPGM followed by conventional PCR detection. We anticipate that use of ddPCR, maximized for sensitivity, will negate the need for extended culture to detect Bartonella and provide physicians with rapid, actionable results within days, rather than weeks. Specific Aim 3: Confirm ddPCR sensitivity using highly concentrated blood samples. One of the significant advantages of ddPCR over standard PCR is the reduction of amplification inhibition by foreign substances, which includes the inherently high amounts of competing host DNA in patient samples that are known to decrease the sensitivity of conventional PCR for low-level infections. Through this aim, we will establish ddPCR sensitivity using concentrated DNA from blood samples that we believe will further circumvent the need for a blood culture to achieve a positive PCR result. Development of this rapid, highly sensitive and specific bartonellosis clinical diagnostic tool will result in an actionable, cost-effective diagnostic assay for physicians. At the current time, there are no SBIR funded projects addressing this gap in the bartonellosis diagnostic space